A recent study demonstrated that rat DNA polymerase ␤ (␤-pol) releases 5 -deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the aminoterminal, 8-kDa domain of ␤-pol and occurs via ␤-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699 -702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap ␤-pol on preincised apurinic/ apyrimidinic DNA using NaBH 4 as the reducing agent. Both 8-kDa domain-DNA and intact ␤-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the ␤-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.Vertebrate DNA polymerase ␤ (␤-pol), 1 a constitutively-expressed monomeric protein ranging from 39 to 45 kDa, has been implicated in DNA repair. Specifically, in vitro experiments indicate that ␤-pol can perform two of the five reactions involved in base excision repair, excision of a 5Ј-terminal dRP from a preincised AP site (1) and DNA synthesis to fill the single-nucleotide gap (2-5). The overall pathway for base excision repair that has been initiated by simple N-glycosylases (glycosylases lacking a concomitant AP lyase activity) is believed to proceed according to the following scheme. (i) A damage-specific DNA N-glycosylase creates an AP site by cleaving an N-C glycosyl bond, thereby releasing a modified base; (ii) a class II AP endonuclease incises the DNA 5Ј to the AP site; (iii) ␤-pol or another dRpase excises the 5Ј dRP terminus to leave a single-nucleotide gap; (iv) ␤-pol or another polymerase fills in the single-nucleotide gap; and (v) DNA ligase seals the gap (6). 5Ј-Terminal dRP might eventually undergo an uncatalyzed ␤-elimination reaction to generate a single-nucleotide gap (half-life Ϸ 2 h) (7), but this reaction is probably too slow to be relied upon in vivo. Coordination of steps (iii) and (iv) by ␤-pol presumably would lead to more efficient DNA repair. Thus, reaction (iii) assures an adequate substrate for ␤-pol, an enzyme without an intrinsic 5Ј-exonuclease activity.Proteolysis studies have revealed a two-domain structure for rat ␤-pol; independent digestions with four proteases resulted in the cleavage of the enzyme within a protease-sensitive region between residues 82 and 86 (8). Treatment with trypsin, for example, yielded an 8-kDa amino-terminal fragment (residues 3-75) and a 31-kDa carboxyl-terminal fragment (residues 87-334). In addition to its recently discovered dRpase activity, the 8-kDa domain (residues 1-87) is known to bind singlestranded DNA with an affinity similar to that of the intact enzyme (8). The 31-kDa domain (residues 88 -334) binds double-stranded DNA but not single-strand...

Objective The aim of this study was to evaluate the safety and efficacy of black cohosh and red clover compared with placebo for the relief of menopausal vasomotor symptoms. Design This study was a randomized, four-arm, double-blind clinical trial of standardized black cohosh, red clover, placebo and 0.625 mg conjugated equine estrogens plus 2.5 mg medroxyprogesterone acetate (CEE/MPA; n = 89). Primary outcome measures were reduction in vasomotor symptoms (hot flashes and night sweats) by black cohosh and red clover compared with placebo; secondary outcomes included safety evaluation, reduction of somatic symptoms, relief of sexual dysfunction, and overall improvement in quality of life. Results Reductions in number of vasomotor symptoms after 12-month intervention were as follows: black cohosh (34%), red clover (57%), placebo (63%), and CEE/MPA (94%), with only CEE/MPA differing significantly from placebo. Black cohosh and red clover did not significantly reduce the frequency of vasomotor symptoms as compared with placebo. Secondary measures indicated that both botanicals were safe as administered. In general, there were no improvements in other menopausal symptoms. Conclusions Compared with placebo, black cohosh and red clover did not reduce the number of vasomotor symptoms. Safety monitoring indicated that chemically and biologically standardized extracts of black cohosh and red clover were safe during daily administration for 12 months.

Extracts of the rhizome of black cohosh [Actaea racemosa L., formerly called Cimicifuga racemosa (L.) Nutt.] were evaluated for potential mechanisms of action in the alleviation of menopausal hot flashes. Ovariectomized Sprague-Dawley rats were administered a 40% 2-propanol extract of black cohosh [4, 40, and 400 mg/(kg.day)] by gavage for 2 weeks with or without estradiol [50 microg/(kg.day)] to determine if black cohosh could act as an estrogen or antiestrogen on the basis of an increase in uterine weight or vaginal cellular cornification. No effects were observed on uterine weight or on vaginal cellular cornification in rats treated with black cohosh alone or in combination with 17beta-estradiol, indicating this black cohosh extract had no estrogenic or antiestrogenic properties in the ovariectomized rat model. To evaluate other potential pathways by which black cohosh might reduce menopausal hot flashes, serotonin activity was first assessed by the inhibition of radioligand binding to cell membrane preparations containing recombinant human serotonin receptor (5-HT) subtypes. A 40% 2-propanol extract of black cohosh was tested against 10 subtypes of the serotonin receptor, revealing the presence of compounds with strong binding to the 5-HT(1A), 5-HT(1D), and 5-HT(7) subtypes. Subsequent binding studies were carried out using 5-HT(1A) and 5-HT(7) receptors because of their association with the hypothalamus, which has been implicated in the generation of hot flashes. The black cohosh 40% 2-propanol extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(7) receptor (IC(50) = 2.4 +/- 0.4 microg/mL) with greater potency than binding of [(3)H]-8-hydroxy-2-(di-N-propylamino)tetralin to the rat 5-HT(1A) receptor (IC(50) = 13.9 +/- 0.6 microg/mL). Analysis of ligand binding data indicated that components of a black cohosh methanol extract functioned as a mixed competitive ligand of the 5-HT(7) receptor. In addition, a black cohosh methanol extract elevated cAMP levels in 293T-5-HT(7)-transfected HEK cells, suggesting the extract acted as a partial agonist at the receptor. The elevation in cAMP mediated by the black cohosh extract could be reversed in the presence of the antagonist methiothepin, indicating a receptor-mediated process. These data suggest that reductions in hot flashes in some women taking black cohosh may not be due to estrogenic properties. This study identifies other possible biological targets of black cohosh that could account for reported biological effects.

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Red clover (Trifolium pratense L., Fabaceae) botanical dietary supplements have received much attention recently for their potential use in the treatment of menopause symptoms, maintenance/improvement of bone and cardiovascular health, and reported benign effects on the breast and endometrium. Literature searches of four computerized databases were run to identify clinical studies of red clover botanical dietary supplements. The manufacturer of the red clover products used in the majority of the studies was contacted for unpublished information and/or clarification regarding the chemical content of their products. Red clover studies were reviewed that pertained to women's health or menopause. Clinical evidence is presently lacking to support the efficacy of semipurified red clover isoflavone extracts for alleviation of climacteric vasomotor symptoms or reduction of low-density lipoprotein levels in the blood. Furthermore, the safety of use of red clover isoflavone supplements in patients with breast or endometrial cancer has not been established. Limited evidence suggests possible efficacy in maintenance of bone health and improvement of arterial compliance, a risk factor for atherosclerosis. This literature review covers red clover botanical dietary supplement clinical studies having a possible impact on the health care of mature and menopausal women, and provides historical perspective regarding the traditional uses of red clover.